Project Summary Textbooks feature an elegant depiction of the human cell, displayed as a phospholipid bilayer-enclosed sphere divided into well demarcated membrane-bound structures called organelles. These micro-reactors, which concentrate and segregate biochemical processes, are typically represented as homogenous compartments. In reality, however, cellular organelles feature substantial sub-organization with individual sub-compartments lacking strict physical barriers. Of particular complexity is the cell nucleus, which orchestrates the reactions that allow transfer of information from a genetic code (DNA) to the functional workhorses of the cell (RNA and protein). Over the past decade, a paradigm shift has occurred in how biologists view nuclear organization. Remarkably, sub-nuclear membraneless organelles (condensates), which include nucleoli and nuclear speckles, form by a physical process called liquid-liquid phase separation (LLPS). The LLPS model posits that condensate assembly is driven by preferential but weak interactions between RNAs and proteins, a process analogous to oil molecules forming droplets in a bowl of vinegar. Despite a flurry of recent papers describing LLPS of diverse protein and RNA complexes in the nucleus, a nuanced understanding of the biophysical and cellular rules governing organization of this cellular emulsion remain poorly understood. For example, systematic studies of the molecular composition and functional significance of nuclear speckles, a multi-phase condensate with peripheral and core components that features numerous splicing factors, are lacking. To begin to address this problem, we mined the human proteome for proteins enriched in serine-arginine (SR) dipeptides, based on the assumption that proteins essential for nuclear speckle formation contain this putative targeting signal. We used CRISPR-Cas9 to individually knockout these proteins in human cells and examined speckle morphology based on several markers. Preliminary data suggests that knockout of many SR proteins, but not other essential genes, results in speckle rounding/swelling (or ?demixing?). One knockout was unique in causing dissolution of speckle cores and collapse of peripheral speckles. We further observed that speckle core dissolution is associated with acute growth defects. However, long-term culture allowed us to generate viable clonal lines lacking these structures. The functional implications of this remain to be tested. The current proposal aims to (1) Quantitatively assess the effect of SR protein knockout on speckle morphology using CRISPR-Cas9 and super-resolution imaging; (2); Define principles of speckle sub-phase partitioning by integrating proximity-based labeling and proteomics with a novel optogenetic platform that triggers demixing of multi-phase speckles; (3) Elucidate the functional consequences of speckle core loss on nuclear architecture and RNA processing using gene loci tracking, RNAseq, and chromosome conformation capture technologies. The results from this project will have vast implications for understanding the function of speckle sub-phases and will inform our understanding of how perturbation of speckle dynamics contributes to disease.